Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching

Significance Circadian clocks adapt us to our rhythmic world, setting the tempo to our lives. Their disruption (e.g., by shiftwork) therefore carries a severe cost in health. The suprachiasmatic nucleus (SCN) is the principal brain clock of mammals, its time keeping pivoting around a delayed negative feedback loop of gene and protein expression. By using “translational switching” as a means to reversibly control the expression of the negative feedback regulator Cryptochrome 1 (CRY1) in SCN organotypic slices, we show that acute changes in the level of CRY1 define circadian time. We thereby bridge theoretical and biochemical perspectives of the SCN clockwork.

detected by a photon multiplier tube (PMT; Hamamatsu, Japan), maintained at 37 °C in a light-tight incubator (CO2 not required as slices were sealed and buffered with HEPES and NaHCO3).
Photon counts were recorded every second and counts were combined in 6-minute bins.

Viral transduction of SCN slices
SCN slices were transduced with AAVs after a medium change (culture medium or recording medium, depending on the experiment) immediately prior to transduction. One μL of AAV with titre of at least 1x1013 GC/mL was dispensed directly on top of the SCN slice and incubated for 7 days before exchanging for fresh medium.

Translational switching
SCN slices were sequentially incubated with each AAV for 7 days separated by a medium change with fresh culture medium. To allow translation of tgCRY1(TAG) the substrate for PylRS, alkene lysine, abbreviated to AlkK, N6-2-propynyloxycarbonyl-l-lysine, (synthesised in-house, stored as 100 mM stock solution made in recording medium, adjusted to pH 7.0) was added to the recording medium with a final concentration of 0.1 -10 mM. Reversibility was confirmed by exchanging fresh recording medium that did not contain AlkK, using 6 washouts for 15 minutes each. In the dose response experiments, AlkK was applied to give a final concentration of either 0, 1, 5 or 10 mM to the double transduced SCN slices.

Confocal time-lapse recordings
Live imaging of SCN organotypic slices was carried out using either Zeiss LSM780 or LSM880 inverted confocal systems, maintained at 37°C. Custom inserts were made to hold up to 6x 35 mm dishes at a time. The "position list" function was used to make simultaneous time-lapse recordings from multiple SCN slices. SCN slices were transferred to 35 mm glass bottom dishes (Mattek) for all live imaging experiments. The same recording medium was used as for the PMT luciferase recordings. For time-lapse recordings, a 10X apochromatic objective was used with the following acquisition parameters: 1024 x 1024 pixel frame size, 4x averaging, 1 frame acquired every 30 minutes for the duration of the experiment.

SCN fixed slices
SCN slices were cut out of their membrane inserts and fixed in 4% PFA in phosphate buffer (as previously) for 30 minutes at room temperature, with gentle shaking. Fixed SCN slices were washed with PBS for 15 minutes, 3 times and mounted on to slides (as previously) with mounting medium with DAPI.

Confocal snapshot imaging
Fixed SCN tissue (sections and slices) were imaged using either Zeiss LSM710, 780 or 880 systems using a 63x oil immersion apochromatic objective. To image the whole coronal view of the SCN, a tile-scan protocol was used within the Zen acquisition software. Particularly in the DAPI channel, this produced some tile artifacts at the joins between tiles, which can be seen in images.
Nonetheless, the tile-scan allowed for higher resolution images of the whole SCN than could be acquired with the highest numerical aperture (NA) 10x objective, an objective that would not have required tiling. Further processing was carried out within FIJI (10).

BioDare
The Fast Fourier Transform -Linear Non-Least Squares (FFT-NLLS) within the BioDare (www.biodare2.ed.ac.uk) circadian rhythms analysis software package (11) was used to analyse rhythmicity of both luciferase and fluorescence-based recordings, where there was stable rhythmicity of at least 5 days. The first 24 hours after any medium change or treatment were excluded from the analysis. The output from software included best-fit period within a circadianrelevant window of 18-32 h periods, relative peak phase, amplitude, goodness of fit (GOF), the latter is an assessment of the robustness of the rhythms. Shapiro-Wilk test of our GOF dataset, in particular the GOF measure of oscillations from all SCN before AAV transduction, ( Figure 2G) showed it to be normally distributed, p = 0.89. An ANOVA is therefore an appropriate test to use when making comparisons between these data.   D. Inferred behavior of limit cycle on withdrawal of ts-CRY1. Confocal imaging showed that signal from ts-CRY1::EGFP was lost within 12 hours of wash-out. Removal of AlkK was also followed by an increase in Cry1 mRNA, which is represented by the dotted trajectory, moving away from the locked state. All SCN re-commenced full amplitude oscillations from the putative CT00. The individual four cycles starting 24 hours after wash-out (mean of five SCN) are plotted in grey, and their overall mean in black, again using PER2 as a proxy for endogenous CRY1.